The three pinene synthases (cyclases) from common sage (Salvia officinalis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins a-pinene, b-pinene, and camphene, in addition to smaller amounts of monocyclic and acyclic monoterpene olefins. In particular, pinene cyclase II produces (-)-a-pinene, (-)-b-pinene, and (-)-camphene competitively at a common catalytic site. These three bicyclic monoterpenes presumably arise by competing proton eliminations and Wagner-Meerwein rearrangement from a common pinyl carbocation intermediate. Despite extensive study, an interesting and important remaining question is the stereochemistry of the terminating CH3 to CH2 elimination of the pinyl intermediate producing the exocyclic isomer, (-)-b-pinene. It is unclear whether the competing endocyclic and exocyclic eliminations are suprafacial or antarafacial. If the OPP- anion is the base that accepts both protons in these eliminations, they should be suprafacial. On the other hand, the occurrence of an antarafacial relationship would require a 2-base mechanism in the competing formation of (-)-a-pinene and (-)-b-pinene. We are addressing this stereochemical question by use of chiral methyl labelling and 3H NMR analysis, allowing us to determine simultaneously the stereochemistry of the CH3 to CH2 eliminations in both b-pinene and camphene biosynthesis. The approach relies on the ability to synthesize [10-3H1, 2H1]-Geranyl Pyrophosphate (GPP) without proton dilution (estimated yields: 2.4 mmol, 56 mCi, 28 Ci/mmol, overall radiochemical yield ~14%). At this scale the enzyme incubation should afford 1-2 mCi of [3H]-b-pinene and [3H]-camphene, judged to be sufficient for 3H-NMR spectroscopy. The chemical shift differences for the two exocyclic vinyl protons are DdH=0.08 ppm for b-pinene and DdH=0.23 for camphene, which should be readily distinguishable in the tritium spectra at 640 MHz. Another interesting stereochemical question arises in the biosynthesis of sabinene hydrate. The cyclopropane ring of this monoterpene is evidently formed by a concerted 1,2-hydride shift and homoallyl participation with inversion of configuration at C-4 if the initial cyclization is assumed to occur as usual in an anti endo conformation of the intermediate (R)-linaloyl PP. The question is whether the 1,2-hydride shift occurs to the re or si face of the a-terpinyl carbocation intermediate, i.e. with minimum or maximum rotation about the exocyclic C-C bond. The direction and magnitude of the rotational movement associated with this 1,2-hydride shift can be elucidated by labelling the trans terminal methyl group of the substrate, i.e. by synthesis of [8-3H1, 2H1]-Geranyl Pyrophosphate (GPP), and isolation of the incubation products for tritium NMR analysis. In a similar fashion, [9-3H1, 2H1]-GPP may be used for investigation of the stereochemistry of the terminal deprotonation in the biosynthesis of limonene, by incubation with limonene synthase. Since theresultant olefinic protons are indistinguishable by proton NMR at 400 MHz, further conversion of the limonene to a nitrosyl chloride derivative will allow direct tritium NMR assessment of the reaction pathway. The initial labelling of these precursors was completed in the latter stages of 1995. Several steps of the subsequent chemistry proved difficult to execute at small scale, and resolution of these problems significantly delayed progress in these projects. Recently, a small sample of product from the limonene synthase study was obtained, but the NMR results were inconclusive. Now that the chemistry is better developed, and cloned enzymes are available in large quantity for all these systems, this project should be able to progress rapidly.